Floral morphology and phenology of Sassafras tzumu (Lauraceae)

Background Sassafras has been considered to belong to trib. Laureae of Lauraceae and has been assumed to have unisexual flowers. However, recent molecular phylogenetic studies have consistently suggested that Sassafras does not belong to the trib. Laureae but to Cinnamomeae and that it is nested within Cinnamomum. A recent morphological study revealed that one of the Asian species, S. randaiense, possesses bisexual flowers that are plesiomorphic in the family Lauraceae. As reports on the flower structure of the second Asian species, S. tzumu, have been contradictory, we wanted to ascertain if it has bisexual flowers or not. If the flowers were bisexual, could earlier reports that they were unisexual have been based on dichogamous flowering? Results In this study, we investigated two populations of S. tzumu. We found that this species has determinate botryoid racemes, and possesses bisexual flowers. Among the three extant species, S. tzumu is more similar to its sister species S. randaiense but markedly different from the American S. albidum: the two Asian species possess bisexual flowers while the American species has unisexual flowers. The bisexual flower of S. tzumu is protogynous, and shows two phenological phases typical of Lauraceae: 1) in a flower, the pistil functions first, the stigma is fresh and white, stamens of the outer two whorls are spreading, anthers do not open, and the staminodes secrete nectar at this stage; 2) in the second phase, the stigma becomes brown, staminodes are withered, stamens of the third whorl stand up and surround the pistil, glands of the third whorl of stamens secrete nectar, and the anthers open and release pollen. Conclusions The similarity of racemose inflorescences between Sassafras and some members of Laureae were caused by parallel evolution; the racemose inflorescence of ancestral Sassafras originated from the thyrsoid-cymose inflorescence in Cinnamomum. The Asian species S. tzumu and S. randaiense possess bisexual flowers with two phenological phases, the American S. albidum evolved unisexual flowers independently from other clades with unisexual flowers in the Lauraceae, i.e., the Laureae, Alseodaphnopsis in the Perseeae and the unisexual clade in the Ocotea complex of the Cinnamomeae.

Sassafras contains three extant species that are disjunctly distributed in East Asia and North America: S. albidum (Nutt.) Nees in North America, S. randaiense (Hayata) Rehder in Taiwan, and S. tzumu (Hemsl.) Hemsl. in mainland China [6]. A previous molecular dating study suggested that the divergence of the North American and Asian species took place in the late Miocene ca. 13.80- 16.69 mya and the divergence between the two Asian species was around 0.61-2.23 mya. The ancestor of Sassafras may have lived in the thermal boreotropical flora of the high paleolatitudes of the Northern Hemisphere in the early Tertiary, and the modern disjunct distribution of Sassafras may have been caused by climate change from the Miocene to the late Neogene [6].
In the past, the three species of Sassafras have been classified in one, two, or three genera. Linnaeus [7] first recognized and described Laurus sassafras L. from North America. This species was considered a distinct genus by Presl [8], who consequently established Sassafras. Nees & Ebermaier [9] transferred Laurus sassafras to Sassafras and gave it a new name, Sassafras officinale T. Nees & C.H. Eberm. However, an earlier name, Laurus albida Nutt., is available for the North American species [10]; Nees [11] transferred it to Sassafras, establishing the currently accepted name S. albidum. Hemsley [12] described one of the Asian species under two different names in the same paper, as Lindera tzumu Hemsl., based on fruiting specimens, and as Litsea laxiflora Hemsl., based on flowering specimens (with racemose-corymbose inflorescences and 4-locular anthers). Later he placed the two species in Sassafras and treated Sassafras tzumu as the accepted name, with Litsea laxiflora as a synonym [13,14]. Lecomte [15] examined flowers of S. tzumu and described it as having bisexual flowers, in contrast to the type species S. albidum. Therefore, he established a new genus, Pseudosassafras Lecomte, to contain only Pseudosassafras tzumu (Hemsl.) Lecomte. Hayata [16] described the second Asian species as Lindera randaiensis, based on a specimen with racemose inflorescences arranged in umbellate clusters at the tip of the branches and 2-locular anthers. Rehder [17] thought that three species were so similar in their vegetative characters (habit, bark, winter buds, deciduous leaves with a tendency toward lobing, inflorescence, and fruit) that they should be placed in a single genus. He thus classified all three species in Sassafras, and transferred L. randaiensis to Sassafras, as Sassafras randaiense. As the number of pollen sacs per anther traditionally had been considered as an important character for the separation of genera in the Lauraceae, Kamikoti [18] established a new genus, Yushunia Kamik., for the only species with two-locular anthers, calling it Yushunia randaiensis (Hayata) Kamik. Most of the other later authors, however, followed Rehder in treating the three species as a single genus [1,2,4,6,[19][20][21][22], in spite of their differences in floral structure.
Different opinions were put forward on relationships of the genus Sassafras. Traditionally Sassafras was ascribed to trib. Laureae because inflorescences of the genus have been considered as racemose with an involucre or frondose transitional leaves at the base, and probably close to Actinodaphne [2,3], or to subtribe Cinnamomineae of trib. Cinnamomeae because of inflorescences lacking involucral bracts, anthers 4-locular, and fruit base embedded in a cupule [1]. A detailed observation of Sassafras randaiense suggested that the inflorescence is a determinate botryoid raceme, and young inflorescences are enclosed within a winter bud by four to six decussate bracts [19]. Recent phylogenetic studies have suggested that Sassafras is sister to Cinnamomum sect. Camphora Meisn. but not to Actinodaphne [4,5]. To understand the evolution of the genus better, the inflorescences of the second Asian species, Sassafras tzumu, are re-examined here, in order to clarify the general pattern of inflorescence structure in the genus.
Flower sex distribution is variable within in the genus. The American Sassafras albidum possesses unisexual flowers, which are somewhat variable in their structure. Male flowers were described as having (almost) no pistillode by Nees [11] ("Pistilli rudimentum nullum" in the genus description, "Pistilli vix ulla vestigia" in the species description), whereas van der Werff [20] mentioned that the terminal flower can have a pistillode. Female flowers were described as having six staminodes by Nuttall [10], as many stamens as in the male flowers or fewer by Nees [11], while van der Werff [20] wrote "staminodes absent or present" in the genus description and "staminodes 6" in the species description. Both Hemsley [12] and Hayata [16] considered the flowers to be unisexual when they described the Asian species; Kamikoti [18] and Keng [21] followed their opinion that the flowers of Sassafras are unisexual. Lecomte [15,23] thought that S. tzumu possesses bisexual flowers. Gamble & Wilson [24] believed that the flowers of S. tzumu were polygamo-dioecious. Li et al. [22] described the genus having unisexual or bisexual flowers; they described male and female flowers separately. Chung et al. [19] observed 20 trees of Sassafras randaiense and finally determined that the flower of the Taiwan species is bisexual, not unisexual. It remains ambiguous whether S. tzumu possesses unisexual flowers, or bisexual flowers, or both.
This study is to examine the inflorescences and flowers of the Asian Sassafras tzumu and compare it with the American S. albidum and the Asian S. randaiense to better understand the morphological diversity and evolution of reproductive characters in the genus.

General morphology
Plants of Sassafras tzumu are tall deciduous trees (Fig. 1A, B). The species blooms from mid-February to early March in Nanjing (Fg. 1A). Leaves are usually trilobed and tripliveined (Fig. 1C, D). Inflorescences occur earlier than leaves. They are pseudoterminal, with a number of racemes clustered at the top of branches and subtended by a few decussate bud scales (interchangeable with 'involucral bracts' in Kostermans [1]) (Fig. 1E). Fruits are globose and seated on top of swollen pedicles; tepals are deciduous or rarely persistent (Fig. 1F).

Inflorescence morphology
The species is deciduous and flowers occur before foliation. Inflorescences of Sassafras tzumu are developed from large terminal perulate buds. Buds are up to 1 cm long before they sprout; bud scales are decussate, With the sprouting of buds, the outermost bud scales (or involucral bracts) fall off and only 4-6 more elongate inner bud scales remain (Fig. 2 B). Inflorescences are pseudoterminal; on average seven inflorescences are clustered below the true vegetative terminal buds, which will elongate and develop into normal vegetative branches (Fig. 2C). Sassafras tzumu has raceme-like but determinate inflorescences (= botryoids) (Figs. 2B, C & E-G). The length of the inflorescence is 4-5 cm. Involucral bracts are at the base of inflorescences and developed from bud scales, and show frondose transitional morphology from rotund to oblong and linear (Fig. 2D). In Sassafras tzumu, each inflorescence has about 11 pedicellate flowers. The position of the flowers on the peduncle is variable. Flowers can be arranged alternately, nearly opposite, or verticillate on the densely brownish pubescent peduncle ( Fig. 2E-G). Rarely a lateral cyme with two flowers is found in place of one flower (Fig. 2G). Pedicles are 4.5-6 mm long, becoming shorter towards the tip. Bracts are linear to filiform, 1-8 mm long, distally becoming shorter.

Flower morphology
Flowers of S. tzumu are obviously bisexual, 7-9 mm in diameter. From the outside inward, the flower consists of six tepals in two whorls, nine fertile stamens in three whorls, one whorl of staminodes and a central fertile  (Fig. 3A, B). Tepals are yellow, lanceolate, slightly obtuse, glabrous, subequal, ca. 3 mm long (Fig. 3C, D). Fertile stamens are inserted on the rim of a very small perianth tube or receptacle; they are subequal, ca. 3 mm long. Anthers are ovoid-oblong, obtuse and emarginate at the apex. Filaments are filiform, longer than anthers, complanate, glabrous (Fig. 3E-L). Stamens of the first and second whorls are eglandular; stamens of the third whorl have a pair of glands at the base of the filaments. Glands are nearly spherical, shortly stipitate ( Fig. 3I-L). Anthers of all observed flowers are 4-locular, with the upper two locules smaller than the lower two locules. All locules of the first and second whorls are ovoidoblong, dehiscing introrsely ( Fig. 3E-H). Lower locules of the third whorl are dehiscing laterally (laterorse); the upper locules are circular, dehiscing apically or slightly introrsely (Fig. 3I-L). Three staminodes alternate with stamens of the third whorl. They are ca. 1.5 mm long, glabrous, with a distinct filament and a ± triangular to somewhat caudate glandular head (Fig. 3M, N). The ovary is ovoid, ca. 1 mm long, superior, positioned in a shallow receptacle. The style is slender, ca. 1.2 mm long. The stigma is white and discoid-dilated (Fig. 3O).

Flower phenology
The bisexual flower of Sassafras tzumu is protogynous. A newly opened flower is functionally female. In this phase, all tepals and stamens are horizontally spreading; stamens are appressed to tepals, and anther locules are closed; the glands of stamens of the third staminal whorl are green; staminodes of the forth whorl become yellow and begin to secrete nectar to attract pollinators; the stigma is white and receptive (Fig. 4A). In the male phase, the stigma becomes brownish; the staminodes turn orange and stop secreting; staminodes and stamens Abbreviations: g, glands; p, pistil; s 1 , the first whorl of stamens; s 2 , the second whorl of stamens; s 3 , the third whorl of stamens; st, staminode; t 1 , the first whorl of tepals; t 2 , the second whorl of tepals. Photos by Zhi Yang of the third whorl bend inward gradually, become upright and enclose the central pistil; staminal glands begin to secrete nectar (Fig. 4B); when staminodes and the third whorl of stamens enclose the pistil, the stamens of the two outer whorls also curve upwards more or less (Fig. 4C); at this time, anther locules open and the pollen is exposed to pollinators (Fig. 4D). Finally, staminal glands stop secreting and turn orange-brown, filaments are relaxed and all stamens curved inwards, the valves of the anther locules become brownish, stamens and style are withered (Fig. 4E, F).

Discussion
Inflorescence types provide important morphological characters for the taxonomy of Lauraceae, especially at suprageneric levels [1][2][3]. According to van der Werff & Richter [3], there are three major types of inflorescences, thyrsoid-paniculate inflorescences lacking an involucre and possessing ultimate cymes having strictly opposite lateral flowers in the trib. Perseeae, thyrsoid-paniculate inflorescences with ultimate cymes having alternate lateral flowers in the trib. Cryptocaryeae, and racemoseumbellate inflorescences in the trib. Laureae. Sassafras possesses botryoid inflorescences (determinate racemes), which originate from axils of scales under a vegetative bud [2] or the axils of leaf organs transitional between bud scales and normal foliage leaves. The genus was classified into the trib. Laureae because its inflorescences were thought to belong to the same type of inflorescences as in other members of the tribe, i.e., Actinodaphne, Laurus, Lindera, Litsea etc. Recent phylogenetic studies have consistently suggested that Sassafras belongs to the trib. Cinnamomeae and is nested within Cinnamomum, while the members of the trib. Laureae form a separate clade [4,5,25]. As a result, it must be assumed that the racemose inflorescences of Sassafras and some members of the trib. Laureae (e.g. Actinodaphne henryi Gamble and A. pilosa (Lour.) Merr.) resulted from parallel evolution, and should not be considered as synapomorphic. The inflorescence of Sassafras is different from those of Lindera and Litsea: 1) the inflorescence of Sassafras is botryoid while the inflorescences of Lindera and Litsea are usually umbellate; 2) the inflorescences of Sassafras originate from the axils of frondose bracts below the terminal bud while the inflorescences of Lindera and Litsea are most commonly inserted on shortened branchlets [1,2]. These differences may corroborate the parallel evolution of similar racemose inflorescences between Sassafras and the trib. Laureae. Kostermans [1] suggested that the involucral bracts (bud scales) of Sassafras are deciduous and drop before anthesis while those of Lindera and Litsea drop after anthesis. However, persistence of involucral bracts is variable in all these genera. We For over a century, flowers of Sassafras had generally been described as unisexual, and anthers of all stamens had been described as introrse [1,22], though Lecomte [15] indicated that S. tzumu possesses bisexual flowers and Gamble & Wilson [24] thought the flowers of S. tzumu were polygamous. Chung et al. [19] observed the flowers of Sassafras randaiense, and found that the species possesses bisexual flowers and anthers of the third staminal whorl are extrorse. Our new observations in this study confirm that S. tzumu has bisexual flowers, too. Contrasting to the Asian species, the American Sassafras albidum has unisexual flowers (Fig. 5). Recent molecular phylogenetic studies have shown that Sassafras is monophyletic, so that there is no need to split it into two or three genera. It is nested within Cinnamomum and the two Asian species S. tzumu and S. randaiense constitute a clade sister to the American species S. albidum [4,5]. As a result, it can be inferred that the ancestor of Sassafras possessed (predominantly) racemose inflorescences and bisexual flowers, and that the two Asian species retained the plesiomorphic bisexual flowers, whereas S. albidum acquired the autapomorphic unisexual flowers after its divergence from the Asian species. Unisexual flowers arose several times in the Lauraceae, e.g. in Alseodaphnopsis in the Perseeae [26], within the Cinnamomeae in the clade including the dioecious species of Ocotea as well as the dioecious genera Endlicheria and Rhodostemonodaphne [27], and in the common ancestor of the tribe Laureae.
Sassafras shows variation in number of anther locules. The American S. albidum mostly has 4-locular anthers [17], but occasionally some of the stamens may have 2-locular anthers (Fig. 5). Hemsley [12] described Litsea laxiflora (= S. tzumu) based on flowering specimens from Hubei (Badong Xian) of China; he ascribed the species to Litsea because the anthers are 4-locular. Hayata [16] described Lindera randaiense (≡S. randaiense) because he noticed that the species has 2-locular anthers. Chung et al. [19] observed variable locule numbers in S. randaiense, i.e., 2-and 4-locular anthers. We observed floral materials from two populations in mainland China, one in Nanjing of Jiangsu Province, and the other in Lushan Botanical Garden of Jiangxi Province. We found only 4-locular anthers and did not see any 2-locular anthers in S. tzumu. We thus confirm the observation of Hemsley [12] that S. tzumu possesses 4-locular anthers. In addition, we found that anthers of the first and second staminal whorl are introrse in S. tzumu, whereas opening of anther locules of the third staminal whorl is variable. The upper two locules are smaller, circular or nearly so, apical and/or slightly introrse, the lower two locules are relatively bigger, ovate to elliptic, and latrorse, not introrse or extrorse, which is different from the extrorse anthers in S. randaiense recorded by Chung et al. [19].
Anther locule number has been considered to be an important taxonomic character in the family Lauraceae. Lindera differs from Litsea in the 2-locular anthers (vs. 4-locular anthers in the latter). Dehaasia differs from Alseodaphne and Nothaphoebe in the 2-locular anther (vs. 4-locular anther in the latter two genera). Plants of the Cryptocarya group usually possess 2-locular anthers (4-locular in Potoxylon Kosterm.). However, 4-locular anthers are considered as the ancestral type [28], and 2-locular anthers in the family are known to have originated from 4-locular anthers multiple times because the genera with 2-locular anthers do not form a clade but belong to a number of separate clades. Sassafras belongs to the trib. Cinnamomeae that shares 4-locular anthers as the plesiomorphic condition, and within the genus Sassafras the number of anther locules changed from four to two in S. randaiense and occasionally in S. albidum. Based on observations in S. albidum (by JGR, pers. observ.), 2-locular anthers in Sassafras originated by reduction of the upper pair of locules. The 2-locular anthers of Aiouea Aubl. and a few Ocotea species resulted by reduction of the upper pair of locules as well [28]. Reduction of the lower pollen sacs is known to occur in Urbanodendron Mez. In the Cryptocarya group, 2-locular anthers arose by lateral fusion of collateral pairs of pollen sacs. However, it remains unclear why the species changed from 4-locular to 2-locular anthers. Parallel evolution of morphological characters is not rare in the family Lauraceae. Besides, plants of Lauraceae are mostly evergreen, but deciduous species occur in Litsea, Lindera, and Sassafras. The deciduous habit of Sassafras originated independently from that in Litsea and Lindera. In addition, a few deciduous species of Lindera possess trilobed leaves, e.g. Lindera obtusiloba. Sassafras has trilobed leaves too, which also originated separately from those in Lindera. Probably the convergent characters led the early authors to ascribe the Asian species of Sassafras to Litsea and Lindera.
In many Lauraceae, flowers are bisexual, e.g. in Perseeae, Cryptocaryeae, and most Cinnamomeae ( Table 1). The bisexual flowers of the family are protogynous and show different phases during pollination [29][30][31][32][33]. In the female phase, the flower open, tepals and all stamens are spreading, the staminodes are yellowish and secrete nectar, the stigma is fresh and receptive [30,31,34]. In the male phase, the stigma becomes withered and the staminodes stop secreting, both stigma and staminodes become reddish brown, stamens of the third whorl stand up and surround the central pistil, the staminal glands of the third androecial whorl secrete nectar, anthers of all stamens open and release pollen [30,31,34]. After pollination, all stamens may come close together and surround the central pistil, and sometimes the tepals come close, too, e.g. in Phoebe chekiangensis C.B. Shang [35] (YY, pers. observ.), but sometimes not, e.g. in Sassafras. In this study, we found that the bisexual flower of S. tzumu performs in a similar way to the typical bisexual flower in Cinnamomum, Persea Mill., Phoebe Nees, and Machilus Nees [30,31,35]. The structure and phenological performance of bisexual flowers facilitate out-crossing in Lauraceae [29,30]. The Asian Sassafras is basically consistent with the general phenology of other bisexual flowers. However, the apical or slightly introrse opening of anther locules of the third staminal whorl implies that self-pollination Yasunia van der Werff trimerous or dimerous bisexual columnar 2 Note: floral characters of genera of Lauraceae extracted mainly from Rohwer [2]; Hypodaphnis was examined by J.G.Rohwer (vouchers: Leeuwenberg 5557, Wilks 1655, Zenker 3033a in HBG); Sinopora is according to Li et al. [36]; Yasunia is from van der Werff & Nishida [37]; Damburneya is from Trofimov et al. [38] may be complementary when out-crossing fails. Further studies are necessary to illuminate how and why the unisexual flowers of S. albidum originated. Though both Sassafras tzumu and S. albidum are deciduous, the former is different from the latter in the flowering time relative to leaf development. Sassafras tzumu flowers earlier than leaf sprouting while S. albidum flowers simultaneously with the flushing of the leaves, with inflorescences originating mainly from the axils of leaf organs that are transitional between bud scales and foliage leaves (pers. observ. by J.G.Rohwer). This furthers our understanding on the divergent evolution of the two species.

Materials and methods
Totally 19 individuals of Sassafras tzumu were observed and sampled in late February and early March of 2022. Three of them were collected from the Lushan Botanical Garden, Jiujiang City, Jiangxi Province, China (28° 51′ 2" N, 115° 46′ 59" E, elev. 142 m), others were collected from Shecun village, Jiangning District of Nanjing, Jiangsu Province, China (32° 0′ 33" N, 118° 56′ 22" E, elev. 118 m). Flowering branches were collected in the field; at least three reproductive shoots were included for each individual. Floral materials were fixed in FAA (mixture of Formalin, Alcohol, and Glacial Acetic Acid). Measurements and observations of flowers were conducted in the Laboratory of Systematic and Evolutionary Botany, Nanjing Forestry University. Photographs were taken using a Nikon D7100 with micro lens (AF Micro-Nikkor 60 mm F2.8D). Detailed morphological observations and photographs were also made under a Stereo Microscope (OLYMPUS SZX10). Voucher specimens of Sassafras tzumu (Y.Yang, Z.Yang & C.Tan QLS-1) were deposited in the Herbarium NF, Nanjing Forestry University; a specimen of S. albidum (Rohwer s.n., 24 Sep 2002) was deposited in the Herbarium HBG, University of Hamburg (Germany). All specimens and materials examined were identified by Yong Yang; pictures of S. albidum were taken in the Botanic Garden of Hamburg (Germany) and identified by Jens G. Rohwer.